Shortened current anti-HCV DAA therapies should be less expensive, but have not provided satisfactory efficacy in nave cirrhotics, treatment experienced non-cirrhotics or GT3-infected patients. Integrating drugs with distinct mechanisms of action (MoA) into DAA regimens could solve this issue. Such drugs are the cyclophilin inhibitors (CypI), which target cyclophilin A (CypA) and which have shown high efficacy in patients. Because of their high barrier to resistance and high efficiency against GT3, CypI are ideal as a rescue therapy for patients who have relapsed with DAA resistance-associated variants. Findings presented at 2016 CROI and EASL conferences suggest that current DAA treatments are not going to be the final solution for the treatment of all HCV patients. Specifically, they suggest i) that despite the efficacy of DAAs, virologic failure occur; ii) that DAA treatment results in the reactivation of HBV; and iii) that HCC recurrence occurs in DAA- treated patients. These novel unexpected findings strongly suggest that new agents, such as CypI, with distinct MoAs, are urgently needed. The unexpected HCC recurrence in HCV patients after DAA treatment is of particular interest for this application since we demonstrated that CypI greatly reduce liver fibrosis. Since an ultimate goal is to restore normal liver function, the antifibrotic activity of CypI represents a major benefit for an anti-HCV treatment and emphasizes that including a CypI into a DAA regimen is an attractive option. Despite the fact that NS5Ai and CypI are efficacious, their MoA are poorly understood. Only knowing that NS5Ai and CypI bind to NS5A and CypA, is not sufficient to understand their MoA. It is critical to elucidate how these key classes of inhibitors exert their MoA at a molecular level rather than only at a clinical level. We showed that NS5A and CypA act in concert to form double membrane vesicles (DMVs), which provide sanctuaries for viral factories, and that both NS5Ai and CypI prevent DMV formation. Here, we propose to conduct a new set of experiments aimed at characterizing the MoA of NS5Ai (Aim 1) and CypI (Aim 2) as well as the role of NS5A (Aim 1) and CypA (Aim 2) in DMV formation. The remarkable antifibrotic activity of CypI - distinct from their antiviral activity - emphasizes that the inclusion of a CypI in a DAA regimen is an attractive option. We propose to conduct a set of experiments aimed at defining the MoA of antifibrotic activities of CypI and to analyze the role of Cyps in fibrosis and HCC (Aim 3). The recent finding that DAA trigger recurrence of HCC is a serious warning that current DAA regimens are not flawless and that additional drugs with different MoAs (CypA neutralization) and with attractive properties (anti-HBV and anti-fibrosis properties) such as CypI are needed. This application seeks to characterize i) the antiviral MoA of NS5Ai and CypI; ii) the antifibrotic MoA of CypI; and iii) roles of NS5A and CypA in DMV formation, and virus life cycle. It also seeks to shift the clinical practice paradigm by including CypI into DAA regimens with the ultimate goal of reducing the cost of treatment and of providing new therapeutic properties, which DAAs lack such as blocking HBV and reducing liver damage.